Mitochondrial biogenesis in rabbit articular chondrocytes transferred to culture

Biology of the Cell

Volume 71, Issues 1–2, 1991, Pages 67-72

Abstract

The effect of the switch to aerobic growth conditions was examined in rabbit articular chondrocytes transferred to culture. Spectroscopic analysis of the cytochromes of the respiratory chain shows that only cytochrome $b$ is present in chondrocytes from cartilage, cytochromes $c, c_{1}$ and $a·a_{3}$ being undetectable as compared with the typical spectrum found in a primary cell culture on day 4. Steady state levels of RNA transcripts of nuclear (cytochrome $c$ ) and mitochondrial genes (cytochrome $b$ and cytochrome oxidase subunits II and III) involved in the oxidative metabolism were determined relative to the RNA transcripts of the nuclear gene for glyceraldehyde phosphate dehydrogenase involved in the glycolytic pathway and to mitochondrial ribosomal RNAs. Chondrocytes transferred to culture showed a general increase in the levels of all transcripts, but the effect on mitochondrial transcripts was much greater ( $× 20$ ) than the effect on nuclear transcripts ( $× 3–4$ ). These results show the absence of a coordinate regulation of the expression of mitochondrial and nuclear genes coding for components of the respiratory chain. The increase in mitochondrial DNA triggered by culture conditions does not appear to be sufficient to account for the enhanced transcription. Concomitant with these mitochondrial changes, the level of transcripts for the collagen II gene involved in the differentiation function decreases dramatically (3% of the control on day 3).

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No differences were found between the average fluorescence intensity values of PpIX in synoviocytes of patients with RA and OA. These values were non-significantly higher after incubation with ALA in comparison with ALA-Me at almost all time points. The average fluorescence intensity of PpIX in CE and chondrons was about ten times lower than in synoviocytes. The presence of preparation of hyaluronic acid (HA) significantly enhanced PpIX induction in chondrons versus treatment only with ALA.
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Citation Excerpt :
In this study we defined <1% O2 as anoxia (since true anoxia could not be achieved and we measured an average of 0.7% O2 in our anoxic culture conditions) and hypoxia as 2% O2 since “normoxia” for chondrocytes in vivo is around 5% O2. Ambient values of 21% O2 may therefore represent abnormally high levels of oxygen and lead to potentially inappropriate chondrocyte responses24,42–44. In our study, 5% O2 appeared to result in the least deviation from control values and some parameters, such as GAG and nitrite/nitrate levels in media, were increased over time after culturing at 21% O2.
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Hypoxia (2% O₂) and anoxia (<1% O₂), acidosis (pH 6.2) and 10 ng/ml IL-1β reduced HOAC cell viability and increased GAG media levels. Acidosis and IL-1β increased nitrite/nitrate release, but increases were moderate at 2% O₂ and significantly reduced at <1% O₂. ATP_i was significantly reduced following hypoxia and anoxia and acidosis. At 48 h cellular ROS levels were increased by acidosis and IL-1β but reduced in hypoxia and anoxia. Mitochondrial membrane potential was reduced in low oxygen, acidosis and IL-1β. Anoxia also resulted in intracellular acidosis. GSH/GSSG ratio was reduced in low oxygen conditions, acidosis and IL-1β.
This study shows that oxygen and pH affect elements of the redox system in HOAC including cellular anti-oxidants, mitochondrial membrane potential and ROS levels.
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Although the majority of the adenosine triphosphate (ATP) in chondrocytes is made by glycolysis rather than by oxidative phosphorylation in mitochondria there is evidence to suggest that reactive oxygen species produced by mitochondrial electron transport (ET) help to maintain cellular redox balance in favor of glycolysis. The objective of this study was to test this hypothesis by determining if rotenone, which inhibits ET and blocks oxidant production inhibits glycolytic ATP synthesis.
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Citation Excerpt :
Therefore, the oxygen consumption rate of those cells is exceptionally low. It has even been reported that the mitochondria of chondrocytes in situ lack certain cytochromes that are required for a fully functional electron transport chain44. Cell morphological studies using electron microscopy have shown that the porcine TMJ disc contains an inhomogeneous distribution of a mixed cell population of fibroblast-like cells and chondrocyte-like cells, which are distinct from hyaline cartilage chondrocytes36.
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Citation Excerpt :
Articular chondrocytes contain only a low number of mitochondria19,20. The activity of the enzymes making up the respiratory chain involved in oxidative phosphorylation (complexes I, II, III and IV), which are located on inner mitochondrial membrane, has been found to be low or absent21; only cytochrome b, a subunit of complex III, has been detected by spectroscopy in freshly isolated chondrocytes22. The role of oxygen in cartilage metabolism is not yet understood.
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Primary bovine articular chondrocytes were cultured in alginate beads (3-D) for 4 weeks or in monolayer under 1% and 21% oxygen for up to 9 days and then returned to 3-D culture for up to 4 weeks. Cells were stained to localise cytochrome c oxidase within the cells. Mitochondrial protein content and cytochrome c oxidase enzymatic activity were determined. Expression of cytochrome c oxidase subunits, COXI and COXIV, was assessed by qRT-PCR.
Cytochrome c oxidase staining remained minimal in chondrocytes cultured in alginate for 4 weeks under 21% oxygen. Mitochondrial protein content and cytochrome c oxidase activity increased significantly during 9 days of chondrocyte expansion in monolayer, accompanied by up-regulation of the COXI mitochondrial gene but not the COXIV nuclear-encoded gene. Cytochrome c oxidase staining increased from day 5 of monolayer culture and remained high even after the cells were returned to 3-D culture for 4 weeks.
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Mitochondrial biogenesis in rabbit articular chondrocytes transferred to culture

Abstract

Cell

Cell Differ

Exp Cell Res

J Biol Chem

J Biol Chem

Exp Cell Res

FEBS Lett

Exp Cell Res

J Biol Chem

Biochem Biophys Res Commun

Cell

Dev Biol

J Biol Chem

J Biol Chem

Effect of DNA conformation on the transcription of mitochondrial DNA

Eur J Biochem

Independent regulation of collagen types by chondrocytes during the loss of differentiated function in culture

Cell